The long-term goal of this project is to understand the molecular basis of cilia formation and maintenance in vertebrate photoreceptor cells and how mutations in cilia genes cause retinal degeneration. In vertebrates, the assembly and maintenance of photoreceptor outer segments begins with the formation of a connecting cilium. The connecting cilium contains a microtubule-based axoneme that is anchored to the apical inner segment by a basal body. Cilia formation begins with the docking of basal bodies at the apical surface of the inner segment. Extension and maintenance of the ciliary axoneme requires the bi-directional motility process of Intraflagellar Transport (IFT) to carry proteins from the photoreceptor inner segment and outer segment. Genetic mutations disrupting the assembly, structure, or function of basal bodies and/or cilia result in a spectrum of diseases known as ciliopathies. These multisyndromic disorders often present with retinal degeneration, kidney disease, mental retardation, and polydactyly. In the current application, we will utilize loss-of-function strategies in zebrafish to investigate the mechanisms controlling basal body localization and determine how mutations in cep290 and arl13b, which are causative for Joubert Syndrome and other ciliopathies, lead to retinal degeneration. In Specific Aim 1, we will examine zebrafish carrying null mutations in cep290 and arl13b for retinal phenotypes. We will also test cep290 and arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, IFT genes, and components of the Planar Cell Polarity (PCP) pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. In Specific Aim 2, we will test the hypothesis that the dynactin complex regulates cytoplasmic dynein motors during basal body docking and ciliogenesis by examining zebrafish mutants in the p150 and p50 subunits of dynactin. In Specific Aim 3, we will directly test the hypothesis that the PCP pathway functions in photoreceptors to control the polarized positioning of basal bodies and that defects in PCP signaling can contribute to photoreceptor degeneration. Our preliminary evidence indicates that basal bodies indeed show a highly polarized arrangement within the adult zebrafish retina. We will determine if this patterning exists during development. We will then express dominant-negative forms the core PCP protein Disheveled (Dvl) in photoreceptors and determine if this leads to retinal degeneration. The results of these studies will allow us to study genes that play critical roles during both cilia assembly and maintenance and will identify novel gene candidates for hereditary blindness so that therapies can be developed to prevent vision loss. PUBLIC HEALTH RELEVANCE: In the vertebrate retina, photoreceptor survival depends on the proper formation and maintenance of the connecting cilium and outer segment. The cilium is a complex organelle that is of great clinical importance because dysfunction in cilia assembly or function can lead to retinal degeneration, kidney disorders, mental retardation, situs inversus, polydactyly, and other conditions. An understanding of the mechanisms that control the formation, positioning, and structural integrity of cilia will lead to the development of treatments for ciliary diseases.